Or Picrotoxin-Induced Convulsions in Mice

Indian Journal of Experimental Biology Vol. 45, August 2007, pp. 720-725

Pro-convulsant effect of cefazolin sodium against pentylenetetrazol- or picrotoxin-induced convulsions in mice Kiran Kumar Akula, Ashish Dhir & S K Kulkarni* Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160 014, India Received 9 February 2007; Revised 12 April 2007

Cefazolin injection (3000 mg/kg, iv) in mice showed several behavioral excitations such as wild running, jumping, rolling, and finally undergoing severe convulsions followed by death. It’s lower doses (500-2000 mg/kg, iv) were unable to produce any convulsions or behavioral excitations in mice. However, cefazolin (500 or 1000 mg/kg, i.v.) when administered before different doses of pentylenetetrazol (PTZ; 40 or 60 mg/kg, ip) or picrotoxin (PTX; 4 or 8 mg/kg, ip.), it produced severe tonic-clonic convulsions in mice. The convulsions or behavioral excitations produced by 3000 mg/kg, iv cefazolin was also reversed by different doses of diazepam (0.5-2 mg/kg, ip) further proving the GABAergic modulatory effect of cefazolin. The results conclude the pro-convulsant action of cefazolin on PTZ- or PTX-induced convulsions, and further confirm the clinical reports. Keywords: Cefazolin, Convulsions, Diazepam, GABA, Pentylenetetrazol, Picrotoxin

Cefazolin (3-{[(5-methyl-1,3,4-thiadiazol-2-yl)thio]- not much experimental evidence available regarding methyl}-8-oxo-7-[2-(1H-tetrazol-1-yl) acetamido]-5- the convulsant ability of cefazolin in animal models. thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid), Therefore, the present study has been designed to a first generation parenteral cephalosporin, is the drug explore the possible convulsant ability of various of choice for surgical prophylaxis1,2 and to treat doses of cefazolin after its acute intravenous various staphylococcal or streptococcal infections3,4. administration in mice, and to investigate its pro- Various clinical evidences have indicated the ability convulsant effect on different doses (doses that are of therapeutically important cephalosporin antibiotics either subeffective or produced mild convulsions) of like ceftriaxone, ceftazidime, cefepime to induce non- GABAA antagonist viz. pentylenetetrazol or picro- convulsive status epilepticus in patients with renal toxin and also to examine the involvement of failure5,6, similarly use of cefazolin at higher doses GABAergic mechanism in the cefazolin-induced results in several side-effects, including its ability to seizures by using diazepam, a GABAA receptor produce convulsions. agonist. Many investigations are being carried out to elucidate the convulsive potential and the mechanism Materials and Methods of action of these antibiotics in humans and Animals — Male Albino mice (Laka strain) experimental animals7-13. Previous work on the weighing between 22–30 g bred in Central Animal convulsant action of penicillins and cephalosporins House facility of Panjab University, Chandigarh were attributed to the suppression of γ-amino butyric acid used. The animals were housed under standard (GABA) system by these antibiotics14-16. Conforming laboratory conditions maintained under a natural light with this hypothesis, in vitro studies with cultured and dark cycle and had free access to food and water. neuronal and [3H] GABA binding indicated that β- Animals were acclimatized to laboratory conditions lactams evoke seizure activity by suppression of the before the experiment. Each animal was used only inhibitory post synaptic responses17-19 mediated by once. All the experiments were carried out between GABA in various brain areas11,20. However, there is 0900 and 1500 hrs. The experimental protocols were ______approved by the Institutional Animal Ethics *Correspondent author Committee (IAEC) and conducted according to the Phone: +91-172-2534114, 2541142 Indian National Science Academy Guidelines Fax: +91-172-2541142 (icmr.nic.in/bioethics/INSA_Guidelines.pdf) for the E-mail: [email protected] use and care of experimental animals. AKULA et al.: PRO-CONVULSANT EFFECT OF CEFAZOLIN AND EPILEPSY 721

Drugs and treatment — The following drugs were were expressed as mean ±SE. The data were analyzed used: Cefazolin sodium(Ranbaxy Research Labo- by using One way Analysis of Variance (ANOVA) ratories, Gurgaon, India), diazepam (Ranbaxy followed by Tukey’s test. In all tests, the criterion for Research Laboratories, Gurgaon, India), pentylene- statistical significance was P < 0.05. tetrazol (PTZ; Sigma, St. Louis, MO, USA) and picrotoxin (PTX; Sigma, St. Louis, MO, USA). All Results the drugs were dissolved in 0.9% w/v NaCl. All the 10 Convulsive potential of different doses of cefazolin doses were chosen as per Williams et al. and the (500-3000 mg/kg, iv) in mice — Cefazolin, when preliminary studies carried out in the laboratory. administered intravenously at doses of 3000 mg/kg Different doses were administered intraperitoneally in produced wild running, jumping and rolling a fixed volume of 1 ml/100g body weight 30 min movements followed by severe convulsions in mice before the animals were subjected to test. All the (showed all the phases of convulsions viz. jerks, drugs were administered intraperitoneally except clonus and extensor and finally death). Lower doses cefazolin which was injected intravenously. In combina- (500, 1000 or 2000 mg/kg, iv) failed to induce any tion studies the pre-determined dose of cefazolin was behavioral abnormality or signs of convulsions in administered 5 min before challenging the animals to mice (Fig. 1a-d). Although 2000 mg/kg, iv cefazolin the sub-convulsive doses of either PTZ or PTX. failed to produce any convulsive behaviour or The experimental protocol consists of various mortality in mice in the present study, a dose of 500 groups viz. Group 1: Cefazolin (500-3000 mg/kg, iv) or 1000 mg/kg, iv was selected as sub-effective doses per se; Group 2: pentylenetetrazol (40-80 mg/kg, ip) of cefazolin for further pro-convulsant studies. per se; Group 3: picrotoxin (4-16 mg/kg, ip) per se; Effect of pretreatment of cefazolin (500-2000 Group 4: cefazolin (500 mg/kg, iv) × pentylene- mg/kg, iv) with sub-convulsive/minimum doses of tetrazol (40 or 60 mg/kg, ip); Group 5: cefazolin (500 pentylenetetrazol or picrotoxin in mice — Pentylene- mg/kg, iv) × picrotoxin (4 or 8 mg/kg, ip); Group 6: tetrazol (80 mg/kg, ip) or picrotoxin (16 mg/kg, ip) cefazolin (1000 mg/kg, iv) × pentylenetetrazol (40 or produced severe tonic-clonic convulsions in mice 60 mg/kg, ip); Group 7: cefazolin (1000 mg/kg, iv) × followed by 100 % mortality. PTZ (60 mg/kg, ip) or picrotoxin (4 or 8 mg/kg, ip); Group 8: diazepam (0.5, PTX (8 mg/kg, ip) induced mild tonic-clonic 1 or 2 mg/kg, ip) × cefazolin (3000 mg/kg, iv) convulsions with decreased mortality while still lower doses of PTZ (40 mg/kg, ip) or PTX (4 mg/kg, ip) Experimental protocol failed to produce any mortality or observable Cefazolin-induced convulsions in mice — Cefa- behavioral changes (Fig.1a-d). For subsequent pro- zolin at different doses (500-3000 mg/kg) was convulsant studies, a sub-convulsive/ minimum dose administered intravenously through the tail-vein in of PTZ (40 or 60 mg/kg, ip) or PTX (4 or 8 mg/kg, ip) mice. Animals were placed in plexi-glass chamber was selected to observe the convulsive potential of and observed for a period of 30 min post cefazolin cefazolin. administration. The parameters noted were mean The sub-convulsive dose of cefazolin (500 or 1000 onset time of jerks, clonus, extensor, and % mortality mg/kg, iv) when injected 5 min before the admini- due to cefazolin. stration of sub-convulsive/minimum doses of PTZ (40 Pentylenetetrazol- and picrotoxin-induced convul- or 60 mg/kg, ip) or PTX (4 or 8 mg/kg, ip), enhanced sions in mice — Pentylenetetrazol (40-80 mg/kg) and the convulsant ability of the later drugs as shown by picrotoxin (4-16 mg/kg) were injected intraperi- decreased onset time of jerks, clonus and extensor toneally to mice. Animals were placed in plexi-glass phase (Figs 2a-c and 3a-c). There was significant chamber and observed for a period of 30 min post increase in mortality in the combination group as PTZ or PTX administration. The parameters noted compared to either drug per se (Figs. 2d and 3d). were mean onset time of jerks, clonus, extensor, and Effect of diazepam (0.5-2 mg/kg, ip) on cefazolin % mortality due to PTZ21,22. (3000 mg/kg, iv)-induced convulsions in mice — Cefazolin (3000 mg/kg, iv) induced behavioral Statistical analysis — One specific group of mice excitation such as wild running, jumping and rolling was assigned to one specific drug treatment condition movements (data not shown), severe convulsions and and each group comprised 6–10 mice. All the values

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Fig. 2 — Pro-convulsant action of cefazolin combined with PTZ Fig. 1 — Effect of cefazolin (Cef, 500-3000 mg/kg, iv), or PTX on the mean onset time of (a) jerks, (b) clonus, (c) pentylenetetrazol (PTZ, 40-80 mg/kg, ip) or picrotoxin (PTX, extensor and (d) mortality in mice. In the combination study, 4-16 mg/kg, ip) on the mean onset time of (a) jerks, (b) clonus, (c) cefazolin (Cef, 500 mg/kg, iv) was administered 5 min before extensor and (d) mortality in mice. P values: < 0.05; as compared challenging the animals to different doses of either PTZ (40 or 60 to acefazolin (500, 1000 and 2000 mg/kg, iv) treated groups; bPTZ mg/kg, ip) or PTX (4 or 8 mg/kg, ip). P values: < 0.05; as (40 mg/kg, ip); c PTZ (60 mg/kg, ip); and dPTX (4 mg/kg, ip) compared to acefazolin treated group; bPTZ treated group; and ANOVA followed by Tukey’s test. cPTX treated group; ANOVA followed by Tukey’s test. death were inhibited by diazepam (0.5-2 mg/kg, ip) various β-lactam antibiotics produces seizures in (Fig. 4a-d). various animal models of epilepsy9,23,24. The Discussion epileptogenic properties of these antibiotics, like their Cefazolin, a first generation parenteral cephalo- antimicrobial properties, are dependent on the sporin1,2 belongs to β-lactam class of antibiotics with integrity of the β-lactam bond25. The tetrazole nucleus a wide spread use for the treatment of various at position 7 of the cefazolin shows a marked bacterial infections3,4. Systemic administration of chemical similarity to pentylenetetrazol26 and

AKULA et al.: PRO-CONVULSANT EFFECT OF CEFAZOLIN AND EPILEPSY 723

Fig. 3 — Pro-convulsant action of cefazolin combined with PTZ or PTX on the mean onset time of (a) jerks, (b) clonus, (c) Fig. 4 — Effect of diazepam (0.5-2 mg/kg, ip) on the mean onset extensor and (d) mortality in mice. In the combination study, time of (a) jerks, (b) clonus, (c) extensor and (d) mortality cefazolin (Cef, 1000 mg/kg, iv) was administered 5 min before produced by cefazolin (3000 mg/kg i.v.) in mice. P values: < 0.05; a challenging the animals to different doses of either PTZ (40 or 60 as compared to vehicle treated control group; ANOVA followed mg/kg, ip) or PTX (4 or 8 mg/kg, ip). P values: < 0.05; as by Tukey’s test. compared to acefazolin treated group; bPTZ treated group; and c PTX treated group; ANOVA followed by Tukey’s test. 33 with distinct domains of the GABAA receptors and block GABA-induced Cl- currents34-37. Cefazolin therefore, the present results with cefazolin confirm showed binding affinity to GABAA receptors in the implications on its role in producing convulsions. concentration-dependent and competitive manners38 Various studies provide strong evidences that and also inhibited [3H] GABA binding to mouse cefazolin has similar intrinsic convulsive activity to synaptic membranes and induced convulsions in a other penicillins27-29. While elucidating the potential concentration dependent manner24. convulsive risk of this antibiotic in clinical use, In the present study, cefazolin per se at higher dose emphasis should be given to its convulsive activity in demonstrated all the parameters of convulsions in experimental animal models. Pentylenetetrazol or mice supporting the clinical reports regarding the picrotoxin-induced seizures are well accepted animal ability of cephalosporins to produce seizures in model of epilepsy involving GABAA antagonistic hospitalized patients receiving continuous iv infusion property22,30-32. Pentylenetetrazol or picrotoxin interact of this class of drugs39,40.

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44 The study used iv route to administer the pam is a positive modulator of GABAA receptors toxic/convulsive dose of cefazolin in order to improve which inhibits the seizure frequency and severity by - the bioavailability and thus increasing the amount of increasing the GABAA receptor mediated Cl the drug penetrating blood brain barrier. At normal conductance32. In the present study, the convulsant concentration cefazolin does not cross the blood brain effect of cefazolin was inhibited by diazepam further barrier and so the amount of the drug reaching indicating that cefazolin induced seizures were due to cerebrospinal fluid (CSF) is less3 but when it is negative GABAergic modulation. administered in massive doses through the intra- venous route, it is assumed that the drug concentration References in CSF is in “instantaneous” distribution equilibrium 1 Oishi C S, Carrion W V & Hoaglund F T, Use of parenteral with that at the site of neurotoxic action, whereas prophylactic antibiotics in clean orthopaedic surgery. A review of the literature, Clin Orthop Relat Res, 296 (1993) there is an initial disequilibrium between the 249. concentration either in serum or in the whole brain 2 De Lalla F, Perioperative antibiotic prophylaxis: a critical and at the site of action41. In the present study, review, Surg Infect (Larchmt), 7 Suppl 2 (2006) S37. cefazolin did not produce any convulsive behaviour at 3 Henry F Chambers, Beta-Lactam antibiotics & other least in normal conscious mice up to 2000 mg/kg, iv inhibitors of cell wall synthesis, in Basic & clinical (Fig.1a-d). This is in accordance with the fact that its pharmacology, edited by Betram G Katzung (Lange Medical 42,43 Books/McGraw-Hill, New York) 2001, 762. penetration into the brain is quite low in animals . 4 Cox V C & Zed P J, Once-daily cefazolin & probenecid for Cefazolin did not produce signs of toxicity or skin and soft tissue infections, Ann Pharmacother, 38 (2004) convulsive behaviour until lethal levels were 458. approached at doses of approximately 2500 to 3333 5 Martı´nez-Rodrı´guez J E, Barriga F J, Joan Santamaria, mg/kg., i.v. in mice (data not shown). Further, the Alejandro Iranzo, Juan A. Pareja, Marian Revilla & Carmen R dela Rosa, Nonconvulsive Status Epilepticus Associated sub-convulsive doses of cefazolin (500 or 1000 mg/kg with Cephalosporins in Patients with Renal Failure, Am J i.v.) when combined with the sub-convulsive/ Med, 111 (2001) 115. minimum doses of either PTZ or PTX, showed an 6 Jos´e Luis Fern´andez-Torre, Mari´an Mart´ınez-Mart´ınez, augmented response with these convulsive drugs and Jes´us Gonz´alez-Rato, Iratxe Maestro, Itz´ıar Alonso, this attributed its potential to enhance the effect of Emilio Rodrigo & Juan Pablo Horcajada, Cephalosporin- various GABA antagonists and to reduce the induced Nonconvulsive Status Epilepticus: Clinical and A Electroencephalographic Features, Epilepsia, 46 (2005) inhibitory GABAergic neurotransmission. The 1550. involvement of N-methyl D-aspartate (NMDA) rece- 7 Yost R L, Lee J D & O'Leary J P, Convulsions associated ptors in cefazolin induced seizures cannot be ruled out with sodium cefazolin: a case report, Am Surg, 43 (1977) 417. but convincing evidences are in favour of its GABAA antagonistic property38. 8 Garretson H D, Reid K H, Shields C B & Noonan J D, The In the present study, it was shown that cefazolin at effect of topical application of antibiotics on the cerebral cortex: an experimental update, J Neurosurg, 50 (1979) 792. higher doses produced convulsions in mice and the 9 Kamei C, Sunami A & Tasaka K, Effects of certain subeffective dose of it potentiates the convulsant Antiepileptics on Cephaloridine- and Cefazolin-induced ability of PTZ or PTX, the GABAA antagonists. This Seizures in Rats, Arch Int Pharmacodyn, 264 (1983) 220. effect clearly supports the hypothesis that cefazolin 10 Williams P D, Bennett D B & Comereski C R, Animal also competitively inhibits GABA-induced Cl- Model for Evaluating the Convulsive Liability of β-Lactam currents by binding directly to the receptor and Antibiotics, Antimicrob Agents Chemother, 32 (1988) 758. 11 Twyman R E, Richard M G & Robert L Macdonald, consequently results in the inhibition of GABAA receptor-mediated inhibitory response. Kinetics of open channel block by penicillin of single GABAA receptor channels from mouse spinal cord neurons Various experimental evidences support the in culture, J Physiol, 445 (1992) 97. involvement of γ-amino butyric acid (GABA) in 12 Yamazaki S, Mochozuki Y, Terai T, Sugimoto M, Uchida I, seizures. GABA is the principal inhibitory neuro- Matsuoka N & Mutoh S, Intracerebroventricular injection of transmitter which attenuates the seizures by binding to the antibiotic cefoselis produces convulsions in mice via GABA receptors involved in activating the inhibitory inhibition of GABA receptors, Pharmacology Biochemistry cascade in different CNS regions and thus plays a Behavior, 74 (2002) 53. ubiquitous role in reducing the excitatory tone. 13 Harika A, Altun O & Nese K B, Cefepime-induced non- convulsive status epilepticus in a peritoneal dialysis patient, Numerous natural and synthetic compounds bind to Pediatr Nephrol, 19 (2004) 445. different modulatory sites of GABA receptors. Diaze-

AKULA et al.: PRO-CONVULSANT EFFECT OF CEFAZOLIN AND EPILEPSY 725

14 De Boer T, Stoof J C & Van Duyn H, Effect of penicillin on 30 Kulkarni S K, Actions of clonidine on convulsions and transmitter release from rat cortical tissue, Brain Res, 192 behaviour. Arch Int Pharmacodyn Ther, 252 (1981) 124. (1980) 296. 31 Fisher R S, Animal models of epilepsies, Brain Res Rev, 14 15 De Sarro A, De Sarro G B, Ascioti C & Nistico G, (1989) 245. Epileptogenic activity of some beta-lactam derivatives: 32 Dhir A, Naidu P S & Kulkarni S K, Effect of structure-activity relationship, Neuropharmacology, 28 cyclooxygenase inhibitors on pentylenetetrazol (PTZ)- (1989) 359. induced convulsions: Possible mechanism of action, Prog 16 Sugimoto M, Fukami S, Hiroshi K, Yamazaki S, Matsuoka Neuropsychopharmacol Biol Psychiatry, 30 (2006) 1478. N, Uchida I & Mashimo T, The β-lactam antibiotics, 33 Huang R Q, Bell-Horner C L, Dibas M I, Covey D F, Drewe penicillin-G and cefoselis have different mechanisms and J A & Dillon G H, Pentylenetetrazole-induced inhibition of sites of action at GABAA receptors, Br J Pharmacol, 135 recombinant gamma-aminobutyric acid type A (GABA (A)) (2002) 427. receptors: mechanism and site of action, J Pharmacol Exp 17 Meyer H & Prince D, Convulsant actions of penicillin: Ther, 298 (2001) 986. effects on inhibitory mechanism, Brain Res, 53 (1973) 477. 34 Pellmar T C & Wilson W A, Synaptic mechanism of 18 Adam A, Walter E M & Wollert U, Inhibition of GABA and pentylenetetrazole: selectivity for chloride conductance, benzodiazepine receptor binding by penicillins, Neurosci Science, 197 (1977) 912. Lett, 18 (1980) 309. 35 Barker J L, McBurney R N & Mathers D A, Convulsant- 19 Phoebe C & David M, Convulsant doses of penicillin induced depression of amino acid responses in cultured shorten the lifetime of GABA-induced channels in cultured mouse spinal neurons studied under voltage clamp. Br J central neurons, Br J Pharmac, 88 (1986) 541. Pharmacol, 80 (1983) 619. 36 Akaike N, Hattori K, Oomura Y & Carpenter D O, 20 Fujimoto M, Munakata M & Akaike N, Dual mechanisms of Bicuculline and picrotoxin block γ-aminobutyric acid-gated GABA response inhibition by β-lactam antibiotics in the A Cl- conductance by different mechanisms, Experientia, 41 pyramidal neurons of the rat cerebral corex, Br J Pharmacol, (1985) 70. 116 (1995) 3014. 37 Suzanne L Hansen, Bonnie B Sperling & Connie Sa´nchez, 21 Kulkarni S K, Handbook of experimental pharmacology, Anticonvulsant and antiepileptogenic effects of GABAA (Vallabh Prakashan, Delhi) 2005, 133. receptor ligands in pentylenetetrazole-kindled mice, Prog 22 Dhir A, Naidu P S & Kulkarni S K, Effect of Neuropsychopharmacol Biol Psychiatry, 28 (2004) 105. cyclooxygenase-2 (COX-2) inhibitors in various animal 38 Sugimoto M, Uchida I, Mashimo T, Yamazaki S, Hatano K, models (bicuculline, picrotoxin, maximal electroshock- Ikeda F, Mochizuki Y, Terai T & Matsuoka N, Evidence for induced convulsions) of epilepsy with possible mechanism the involvement of GABAA receptor blockade in of action, Indian J Exp Biol, 44 (2006) 286. convulsions induced by cephalosporins, Neurophar- 23 Hartsveldt C V, Petit T L & Isaacson R L, Epileptogenic macology, 45 (2003) 304. effect of several penicillins and penicillin-related 39 Aziza K C MD, Salam I G MD, Saed A, Al-Ghwery MD, compounds in rat neocortex, Epilepsia, 16 (1975) 449. Sulaiman A, Al-Mohaimeed MD & Saad A Alshahwan MD, 24 Shimada J, Hori S, Kanemitsu K, Shoji Y, Nakashio S & Cephalosporin-Induced Nonconvulsive Status Epilepticus in Yanagawa A, A comparative study on the convulsant a Uremic Child, Pediatr Neurol, 30 (2004) 135. activity of carbapenems and beta lactams, Drugs Exp Clin 40 Rama M, David J, Daljeet R & Amit B, Nonconvulsive Res, 18 (1992) 377. status epilepticus due to cefepime in a patient with normal 25 Michael J Gutnick, Hans Van Duijn & Nathan Citri, renal function, Epilepsy & Behavior, 8 (2006) 312. Relative convulsant potencies of structural analogues of 41 Yasuyoshi I, Manashi N & Masato Y, Effect of isoniazid on penicillin, Brain Res, 114 (1976) 139. the pharmacodynamics of cefazolin-induced seizures in rats, 26 De sarro A, Ammendola D, Zappala M, Grasso S & De Drug Metab Pharmacokinet, 20 (2005) 117. sarro G B, Relationship between structure and convulsant 42 Swinyard E A, Brown W C & Goodman L S, Comparative properties of some β-Lactam antibiotics following intra- assays of antiepileptic drugs in mice and rats J Pharmacol cerebroventricular microinjection in rats, Antimicrob Agents Exp Ther, 106 (1952) 319. Chemother, 39 (1995) 232. 43 Kamei C, Sunami A & Tasaka K, Epileptogenic activity of 27 Walker A E & Johnson H C, Convulsive factor in cephalosporins in rats and their structure-activity commercial penicillin, Arch Surg, 50 (1945) 69. relationship, Epilepsia, 24 (1983) 431. 28 Curtis D R, Game C J A, Johnston G A R, McCulloch R M 44 Chan C Y & Farb D H, Modulation of neurotransmitter & Maclachlan R M, Convulsive action of penicillin, Brain action: control of the γ-aminobutyric acid response through Res, 43 (1972) 242. the benzodiazepine receptor, J Neurosci, 5 (1985) 2365. 29 Gerald M C, Massey J & Spadaro D C, Comparative convulsant activity of various penicillins after intracerebral injection in mice, J Pharm Pharmacol, 25 (1973) 104.